We reported previously that insulin is essential for the accumulation of casein mRNA in explants of murine mammary tissue. Since the rate of accumulation of mRNA is a function of both the rate of its transcription and of its half-life, it was of interest to determine which component is influenced by insulin. Others had shown that prolactin, another hormone essential for the accumulation of murine casein mRNA, markedly increases the half-life of the mRNA and increases its transcription rate in the presence of insulin. Our studies confirm the marked effect of prolactin on the half-life of the mRNA, and demonstrate that insulin is not required for this effect. However, we have also observed that prolactin does not stimulate the transcription of the 25K mRNA gene in the absence of insulin. Although insulin has been shown previously to exert a negative influence on the transcription of the phosphoenolpyruvate carboxykinase gene, in liver, the mammary system may be the first known instance in which insulin has been demonstrated to exert an essential, positive influence on transcription. We previously reported that estrogen-depleted renders mouse mammary cells in capable of casein gene expression in the presence of insulin, glucocorticoid and prolactin, These cells retain responsiveness to insulin and glucocorticoid, but lack responsiveness to prolactin. The inability to express casein genes was ascribed to a post-binding prolactin-related lesion. However, our recent results on mammary cells from estrogen-depleted rats cast a different interpretation on the role of estrogen in mammary differentiation. The estrogen-depleted rat cells have lost about 90% of their capacity for casein gene expression. They, too, retain full general insulin - and glucocorticoid - responsiveness. However, unlike the corresponding mouse cells, the estrogen-depleted rat cells also retain general responsiveness to prolactin. In the absence of the female sex steroid, rat mammary cells appear to lose only their unique responsiveness to the lactogenic hormones.